porcine kobuvirus 1 in healthy and diarrheic pigs genetic detection and characterization of virus and co infection with rotavirus a

Infection, Genetics and Evolution 49 (2017) 73–77 Contents lists available at ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid Research paper Porcine kobuvirus in healthy and diarrheic pigs: Genetic detection and characterization of virus and co-infection with rotavirus A Anna Jackova a, Ivan Sliz a, Rene Mandelik a, Slavomira Salamunova a, Jaroslav Novotny a, Mariana Kolesarova b, Michaela Vlasakova a, Stefan Vilcek a,⁎ a b University of Veterinary Medicine and Pharmacy, Komenskeho 73, SK-041 81 Kosice, Slovakia Faculty of Science, P.J Safarik University, Srobarova 2, SK-041 80 Kosice, Slovakia a r t i c l e i n f o Article history: Received September 2016 Received in revised form January 2017 Accepted January 2017 Available online 10 January 2017 Keywords: Porcine kobuvirus Rotavirus Diarrhea Phylogenetic analysis a b s t r a c t The porcine kobuvirus (PKV-1) is believed to be an enteric virus To investigate the prevalence of PKV-1 in pigs, virus was detected by RT-PCR in rectal swabs originating from 414 healthy and diarrheic pigs of different age categories on farms in Slovakia Among all ages of animals, PKV-1 was detected equally in diarrheic (63.8%) and clinically healthy (62.9%) pigs PKV-1 was more often detected in diarrheic (74.6%) than in healthy (64.4%) suckling piglets (b 28 days) but data was not statistically significant Results in weaned (28–70 days) and fattening (N 70 days) of both healthy and diarrheic pigs were inconsistent ranging in interval 56.2% to 67.9% This study did not confirm a clear relationship of PKV-1 infection with diarrhea in pigs Rotavirus A infection was detected among the same animals in 39% diarrheic and 9.2% healthy suckling piglets (p b 0.001) confirming rotavirus as a causative agent of diarrhea in this age group The difference was not significant in older pigs with both diarrheic and healthy pigs being infected within a range of 0% to 12.2% Co-infection with PKV-1 and rotavirus A was detected overall in 5.6% of healthy and in 13.5% of diarrheic pigs and was highest in suckling piglets (33.9%) The PKV-1sequences from pigs in Slovakia were analyzed at the genetic level in the partial 3D gene region for the first time The viral sequences were grouped in phylogenetic clusters according to their farm of origin When compared with 157 nucleotide sequences originating from pig samples of different countries around the world Slovakian PKV-1 sequences were clustered in the phylogenetic tree with Asian sequences but not with nucleotide sequences from the neighbouring countries of Czech Republic or Hungary © 2017 The Authors Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Introduction The enteric viruses of farm animals are widely studied around the world due to their high economic impact The application of moleculargenetic techniques in recent years has revealed novel viruses the biological role of which is not known yet Recently the family of pig enteric viruses, namely genus Kobuvirus comprising human and bovine kobuvirus, was enlarged with a new member, porcine kobuvirus 1—PKV-1 (Khamrin et al., 2014; Reuter et al., 2011) This virus was described for the first time in faecal samples of healthy pigs originating from Hungary in 2008 (Reuter et al., 2008) Genetic analysis revealed that the porcine kobuvirus belongs to the family Picornaviridae (Reuter et al., 2009) At present, new taxonomical classification for kobuviruses was proposed (http://www picornaviridae.com/kobuvirus/kobuvirus.htm) The genus Kobuvirus ⁎ Corresponding author E-mail addresses: anna.jackova@uvlf.sk (A Jackova), sliz.ivan@gmail.com (I Sliz), rene.mandelik@uvlf.sk (R Mandelik), slavomira.salamunova@uvlf.sk (S Salamunova), jaroslav.novotny@uvlf.sk (J Novotny), mariana.kolesarova@upjs.sk (M Kolesarova), michaela.vlasakova@uvlf.sk (M Vlasakova), vilcek@uvm.sk (S Vilcek) consists of three species, Aichivirus A (formerly Aichi virus), Aichivirus B (formerly Bovine kobuvirus) and Aichivirus C The species Aichivirus C consists of a single type: porcine kobuvirus Kobuviruses are small non-enveloped viruses comprising a singlestranded positive sense oriented RNA genome of 8.2 kb nucleotides in length, excluding a poly(A) tail, and with an organization typical for viruses of the family Picornaviridae Starting from the N-terminus, the virus polyprotein is divided into non-structural protein L, then three structural capsid proteins VP0, VP3 and VP1 followed by seven nonstructural proteins 2A, 2B, 2C, 3A, 3B, 3C, and 3D The genome is flanked by 5'UTR and 3'UTR regions (Reuter et al., 2009; Yu et al., 2011) The RNA-dependent RNA polymerase (3D) is the most conserved genomic region and it is frequently used for the detection of kobuvirus RNA in clinical samples (Wang et al., 2011; Yu et al., 2011) Kobuviruses are distributed in healthy and diarrheic swine worldwide, including Europe (Di Profio et al., 2013; Reuter et al., 2008; Zhou et al., 2016), Asia (Khamrin et al., 2010; Park et al., 2010; Wang et al., 2011; Yu et al., 2009), South America (Barry et al., 2011; Ribeiro et al., 2013), North America (Verma et al., 2013) and East Africa (Amimo et al., 2014) Although kobuviruses have been frequently detected in http://dx.doi.org/10.1016/j.meegid.2017.01.011 1567-1348/© 2017 The Authors Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) 74 A Jackova et al / Infection, Genetics and Evolution 49 (2017) 73–77 enteric samples their role in diarrhea or gastroenteritis is still unknown The situation is complicated by the observation that porcine kobuvirus has been detected in both healthy and diarrheic pigs (Zhou et al., 2016) Kobuvirus infection has been observed with other co-infecting viruses such as PCV2 (Porcine circovirus type 2), astrovirus, rotavirus A, PEDV (Porcine epidemic diarrhea virus), TGEV (Transmissible gastroenteritis virus) (Park et al., 2010; Zhao et al., 2016; Zhou et al., 2016) which complicates the potential pathogenic role of PKV-1 in diarrhea The virus has been detected more often in young animals with diarrhea (Khamrin et al., 2009; Park et al., 2010; Verma et al., 2013) but published results were not always consistent Rotavirus is a major pathogen associated with acute gastroenteritis in young animals and humans The Rotavirus genus is divided into at least genetic groups or serogroups (A–G) Rotavirus A is the most important rotavirus group due to its high prevalence and pathogenicity, including swine (Papp et al., 2013) The aim of this work was to investigate the prevalence of kobuvirus in healthy and diarrheic pigs The rate of co-infection with rotavirus A was also studied to search for a possible association between both infections and diarrhea In addition, porcine kobuvirus positive samples from Slovakia were studied at the genetic level to find the phylogenetic relationship with the worldwide PKV-1 sequences Material and methods 2.1 Samples Rectal swabs were collected using swab applicator (Sarstedt AG&Co, Germany) from 414 pigs of different age from 17 pig farms located in Slovakia Samples were collected from clinically healthy (n = 251) and diarrheic (n = 163) animals which were divided into suckling piglets (animals before weaning b28 days, n = 146), weaned (28–70 days, n = 147) and fattening (N70 days, n = 121) pigs The health status of pigs was observed and evaluated by qualified veterinarians directly on the farm 2.2 Isolation of RNA The swabs were processed in the laboratory by elution into ml of 0.01 mol/l PBS (Merck Millipore Corp., USA) for 30 The eluted solution was vortexed at 2000 rev min−1 for and then centrifuged at 14000 ×g for Total RNA was isolated using TRIzol Reagent (Life Technologies, USA) from 200 μl of rectal eluate according to the manufacturer's instruction and dissolved in 20 μl of molecular grade water (Merck, GmbH, Germany) Samples with aliquots of isolated RNA were stored at −80 °C 2.3 Detection of kobuvirus by RT-PCR The cDNA was synthesized by reverse transcription in a 20 μl reaction mixture comprising μl of isolated RNA, μM of random hexamers (Invitrogen, USA), 0.5 mM dNTPs, 200 U RevertAid Premium reverse transcriptase with × RT buffer (Thermo Fisher Scientific, Inc., USA), 20 U RNase inhibitor (Takara Bio, Inc., Japan) and molecular grade water (Merck, GmbH, Germany) The mixture was incubated at 65 °C for and then chilled on ice to destroy RNA secondary structure Subsequently, the mixture was incubated at 25 °C for 10 min, then at 50 °C for 30 to synthesise cDNA and at 85 °C for to terminate reaction The detection of PKV-1 genome by PCR was based on the amplification of a 495 bp fragment of 3D gene using primers KoVF/KoVR (Yu et al., 2011) The PCR reaction mixture (25 μl) was composed of × ThermoPol reaction buffer (New England Biolabs, Inc., USA), 0.2 mM dNTPs (Thermo Fisher Scientific, Inc., USA), 0.3 μM of each primer, 0.5 U Taq DNA polymerase (New England Biolabs, Inc., USA), μl cDNA and molecular grade water (Merck, GmbH, Germany) The PCR was run with the following thermal profile: cycle at 95 °C for min, and 37 cycles with denaturation at 95 °C for 30 s, annealing at 52 °C for min, and extension at 68 °C for and final extension at 68 °C for The size of PCR products was checked by electrophoresis in 2% agarose gel after staining with GelRed™ (Biotum, Inc., USA) and visualization by Gel Doc EZ imager (Bio-Rad Laboratories, Inc., USA) 2.4 Detection of rotavirus A by RT-PCR The same cDNA as prepared for kobuvirus was used in a PCR for the detection of rotavirus A The single PCR for group A rotavirus was based on the amplification of a 309 bp fragment of VP6 gene using primers rot3 and rot5 (Elschner et al., 2002) The reaction conditions and thermal profile were the same as described for the kobuvirus detection 2.5 Sequencing of DNA and phylogenetic analysis of PKV-1 Twenty PCR amplicons obtained from PKV-1 positive samples originating from selected farms were sequenced in both directions using Sanger's method employing fluorescently labeled ddNTPs The purification of PCR amplicons and sequencing was carried out by a commercial company (Microsynth Austria GmbH, Austria) The selection of PCR amplicons was done from healthy and diarrheic pigs on farms covering entire investigated area The nucleotide sequences were deposited into GenBank under accession numbers KY242652-KY242671 The chromatograms were checked and edited by the computer programme SeqMan The nucleotide sequences were aligned by the computer program MegAlign (Lasergene, DNASTAR, Inc., USA) The phylogenetic tree was constructed by the neighbor-joining method using the Kimura-2 parameter incorporated in the computer package program MEGA6 (Tamura et al., 2013) 2.6 Statistical analysis Statistical analyses of data were performed by chi-square (χ2) test with confidence limits of 95%, P b 0.05 (statistically significant) or 99%, P b 0.01 (highly statistically significant) using GraphPad Prism for Windows (GraphPad Software, USA) Results 3.1 Prevalence of porcine kobuvirus in clinically healthy and diarrheic pigs PKV-1 was detected in all 17 pig farms The results are summarized in Table When analysing pigs of all age categories together, the RT-PCR analysis of 414 enteric samples revealed that 262 pigs (63.3%) were positive for kobuvirus RNA Of the virus positive pigs, 158 (62.9%) were clinically healthy and 104 (63.8%) were diarrheic animals When looking across age categories the kobuvirus RNA was more often detected in diarrheic suckling piglets (74.6%) than in diarrheic weaned (56.2%) and fattening (61.3%) pigs but the differences were not statistically supported (P = 0.086, χ2 = 4.894) The same three Table Prevalence of porcine kobuvirus in healthy and diarrheic pigs Age group Suckling Weaned Fattening Total Number 146 147 121 414 Healthy (n = 251) Diarrheic (n = 163) Total (n = 414) Posit/negat Posit (%) Posit/negat Posit (%) Posit/negat Posit (%) 56/31 50/24 52/38 158/93 64.4 67.6 57.8 62.9 44/15 41/32 19/12 104/59 74.6 56.2 61.3 63.8 100/46 91/56 71/50 262/152 68.5 61.9 58.7 63.3 A Jackova et al / Infection, Genetics and Evolution 49 (2017) 73–77 75 age categories of healthy animals were characterized with values 64.4, 67.6 and 57.8% The analysis of individual animal age categories did not result in big variations either No statistically significant difference between diarrheic (74.6%) and healthy (64.4%) animals were detected in piglets (P = 0.193, χ2 = 1.698) or in the other age categories 3.2 Prevalence of rotavirus A in healthy and diarrheic pigs Rotavirus A was detected in of 17 farms The results are summarized in Table Rotavirus infection was observed in 45 (10.9%) of all 414 pigs analyzed Among diarrheic pigs 28 of 163 (17.2%) animals were positive for rotavirus mostly due to farms with poor management which were highly affected by rotavirus infection In comparison to PKV-1 infection, rotavirus infection was most often detected in diarrheic suckling piglets (39.0%) than in diarrheic weaned (4.1%) or fattening (6.5%) pigs which was statistically highly significant (P b 0.001; χ2 = 30.987) Healthy animals were also partially infected with rotavirus infection (piglets—9.2%; weaned pigs—12.2%) but fattening pigs were not infected at all When comparing diarrheic suckling piglets (39.0%) with healthy animals of the same category (9.2%) a highly significant association of rotavirus A infection with diarrhea was observed (P b 0.001, χ2 = 18.652) The other two age categories did not show a similar phenomenon 3.3 Co-infection PKV-1 with rotavirus A Overall, PKV-1 and rotavirus A co-infection was detected in 13.5% (22/163) of diarrheic and 5.6% (14/251) of healthy pigs Diarrheic suckling piglets were more often co-infected with both viruses 33.9% (20/ 59) than weaned pigs 2.7% (2/73) Co-infection was not observed in fattening pigs 3.4 Genetic analysis of a part of the kobuvirus 3D gene The alignment of the 443 bp long nucleotide sequence fragments of the 3D gene (PCR primers were omitted) prepared from 20 virus samples originating both from healthy and diarrheic pigs did not reveal any insertions or deletions The viral sequences obtained from pig faecal samples collected in Slovakia displayed nucleotide identity of 89.0– 100% to each other, and shared the closest relatedness (86.9–100%) with 157 additional sequences deposited in GenBank originating from PKV-1 identified in different countries around world To find the relationships among kobuvirus sequences, a phylogenetic tree was constructed from nucleotide sequences of a partial region of the 3D gene (Fig 1) The phylogenetic tree indicated that PKV-1 sequences from the pig farms in Slovakia were clustered according to the farm of origin The sequences from Slovakia formed clusters which corresponded to pig farms from which samples were collected Viral sequences originating from the same farm were found not always absolutely identical independently if they originated from healthy or diarrheic animals but this variability did not lead to the their relocation Table Prevalence of porcine group A rotavirus in healthy and diarrheic pigs Age group Suckling Weaned Fattening Total Number 146 147 121 414 Healthy (n = 251) Diarrheic (n = 163) Total (n = 414) Posit/negat Posit (%) Posit/negat Posit (%) Posit/negat Posit (%) 8/79 9/65 0/90 17/234 9.2 12.2 0.0 6.8 23/36 3/70 2/29 28/135 39.0 4.1 6.5 17.2 31/115 12/135 2/119 45/369 21.2 8.2 1.7 10.9 Fig Phylogenetic tree of PKV-1 nucleotide sequences based on a fragment of 3D gene (443 bp) Sequences from Slovakia are in bold print Boxed sequences originate from diarrheic pigs, non-boxed sequences are from healthy animals Other sequences were acquired from GenBank and they are labeled with accession numbers and country of origin 76 A Jackova et al / Infection, Genetics and Evolution 49 (2017) 73–77 into other significant phylogenetic clusters The kobuvirus sequences originating from Slovakia were located in different phylogenetic clusters to PKV-1 from neighbouring Hungary or the Czech Republic Generally, the PKV-1 sequences from Slovakia were clustered more with Asian sequences than with PKV-1 sequences originating from Europe Discussion This work describes for the first time the identification of PKV-1 in pig farms within Slovakia Overall, the prevalence of kobuvirus was similar in healthy and diarrheic pigs Although diarrheic suckling piglets was the most frequently infected group in our study the results were not statistically significantly different from healthy piglets, similar as in two other age categories In contrast rotavirus infection in suckling piglets was significantly associated with diarrhea (Table 1) Historically, PKV-1 was first detected in enteric samples from healthy 10 day old piglets in eastern Hungary when searching for astroviruses (Reuter et al., 2008) This observation was subsequently confirmed by the detection of kobuvirus in 30.1% of healthy pigs in China (Yu et al., 2009) The association of kobuvirus with diarrhea was proposed following the observation that 99% of diarrheic pigs in Thailand were kobuvirus positive (Khamrin et al., 2009) The epidemiological situation in Slovakia and neighbouring countries shows the prevalence of PKV-1 in pigs as 87.3% in Czech Republic (Dufkova et al., 2013), 81% in Hungary (Zhou et al., 2016), 63.3% in Slovakia (this work) and 46% in Austria (Zhou et al., 2016) Healthy (50%) and diarrheic (43.8%) pigs in Austria were similarly infected (Zhou et al., 2016) Our overall values presented in this work were also similar (62.9% for healthy pigs versus 63.8% for diarrheic pigs) A significantly higher prevalence of PKV-1 infection was associated with diarrheic (92.3%) than healthy (54.5%) pigs in Hungary (Zhou et al., 2016) The records on the prevalence of PKV-1 in other European countries have also been variable Higher prevalence in diarrheic (74.4%) than in healthy (47.5%) pigs has been observed in Spain (Zhou et al., 2016) The opposite situation has been observed between diarrheic (17.4%) and healthy (60.8%) animals on Swedish farms (Zhou et al., 2016) Data on the prevalence of PKV-1 from non-European countries are also contradictory PKV-1 has been detected in similar numbers of diarrheic (21.9%) and healthy (21.7%) pigs in USA (Verma et al., 2013) The high density pig population in Asia is also infected with PKV-1 Among healthy pigs in Japan, 45.4% were infected with PKV-1 (Khamrin et al., 2010) Park et al (2010) detected more pigs positive for PKV-1 in diarrheic (97.8%) than in healthy (40.0%) suckling piglets in Korea similar to results from the Shanghai region of China (Wang et al., 2011) However, there is a report from Korea where PKV-1 has been found more often in non-diarrheic (45.5%) than in diarrheic (32.6%) pigs (An et al., 2011) Nearly all diarrheic piglets were positive for PKV-1 in Thailand (Khamrin et al., 2009) In most studies, piglets were more likely to be infected with PKV-1 than older individuals (Park et al., 2010; Reuter et al., 2010; Verma et al., 2013) One other group has reported that post-weaning pigs were more frequently infected with PKV-1 than suckling piglets (Di Bartolo et al., 2015) In this context our results have shown that diarrheic piglets were slightly more likely to be infected with PKV-1 (74.6%) than two older groups of animals (56.2% and 61.3%) (see Table 1) but the data were not significant Since kobuvirus is believed to be an enteric virus we have hypothesized that there could be some disease association due to co-infection with other enteric pathogens such as rotavirus In our study rotavirus infection was significantly less often observed in pigs than kobuvirus infection The diarrheic piglets were the most affected group by rotavirus infection (Table 2) although at lower rate than that observed with PKV1 (Table 1) The healthy animals were less infected by rotavirus and fattening pigs were not infected by rotavirus at all (Table 2) The detection of rotavirus in healthy piglets and weaned pigs could be explained by an early stage of infection before diarrhea has developed The co-infection of PKV-1 with rotavirus was observed in both healthy and diarrheic pigs but the rates of co-infection were low (5.6% of healthy and 13.5% of diarrheic pigs) No doubt that while rotavirus infection results in diarrhea among piglets there is no clear association of kobuvirus infection with diarrhea among piglets in our study, which contrasts with results elsewhere (Khamrin et al., 2009; Park et al., 2010) We realize that diarrhea in pigs is very complex due to possible co-infections with other bacterial, viral and parasitic infections as well as nutritional and husbandry conditions The co-infection of PKV-1 with other viruses has been observed in other laboratories For example, Dufkova et al (2013) found mixed infections in 47.3% of asymptomatic pigs, mostly with astrovirus and sapovirus similar to those observed by Reuter et al (2008) Park et al (2010) detected 81% PKV-1 positive samples in diarrheic pigs, which were co-infected with to other viral and bacterial enteric agents Zhao et al (2016) published that co-infection of PKV-1 with two or more viruses (astrovirus type 4, rotavirus A, PCV2) has been found in 65.0% of healthy and 55.1% of diarrheic pigs but in piglets the co-infection rates have been significantly higher in diarrheic than in healthy animals similar to our work with rotavirus A Diarrheic pigs from China, where PEDV have been detected in 24.2% of animals, the co-infection of PKV-1 with PEDV was more often (50%) but only occasionally with rotavirus (1.1%) or TGEV (1.1%) (Zhao et al., 2016) Findings from around world on the association of PKV-1 infection with diarrhea in pigs are controversial They raise the question of whether kobuvirus is really associated with diarrhea and is a cause of gastroenteritis in pigs The experimental infection of pigs with PKV-1 has not been done yet because the virus is not cultivable In our opinion inconsistent results published by different groups on the variable prevalence of PKV-1 in healthy and diarrheic pigs are due to: i) differences in the evaluation of diarrhea status in pigs and variability in sampling, ii) variation in the methods of viral RNA isolation from clinical samples and PCR assays used for the detection of virus iii) PKV-1 may differ in virulence resulting in variable severity of diarrhea and excretion of virus, iv) PKV-1 has no influence on diarrhea and its pathogenic role in the enteric system is not significant, v) PKV-1 plays an as yet unknown role during viral and bacterial co-infection of pigs which can reduce the health status of pigs resulting in diarrhea We believe that just the top part of the microbiome iceberg of the pig enteric system has been analyzed so far The application of next generation sequencing methods will contribute further and this will be our scientific activity in the near future The PKV-1 nucleotide sequences originating from samples collected in Slovakia were 89–100% identical in the 3D gene, close to the range of 86–100% when they were compared with nucleotide sequences deposited in GenBank The values indicate that the sequences from Slovakia were almost as variable as sequences from the rest of the world This is quite surprising when Slovakia is a small country and most of the sequenced virus samples were collected from farms in a region of around 50 km diameter There is no final theory how to explain this phenomenon but it would suggest an independent evolution of PKV-1 with similar mutation rates in different parts of world Phylogenetic analysis revealed that PKV-1 sequences from Slovakia were clustered according to the farm of sample origin Surprisingly, they were not clustered near sequences originating from neighbouring countries but rather with Asian or African viral sequences (Fig 1) Similar phenomenon has been observed with Italian PKV-1 sequences (Di Profio et al., 2013) At present it is difficult to explain this observation since there is not any known trading of pigs or products from/to Slovakia outside Europe However, it cannot be excluded that when more porcine kobuvirus samples originating from Europe have been sequenced, the shape of the phylogenetic tree may change In our studies viral sequences of the partial 3D gene from healthy and diarrheic pigs have been the same or close related at the genetic level On the other hand, Chinese PKV-1 originating from diarrheic pigs analyzed in the VP1 region have been more genetically diverse A Jackova et al / Infection, Genetics and Evolution 49 (2017) 73–77 than PKV-1 from healthy pigs (Jin et al., 2015) Differences between these two observations may be explained by stronger genetic conservation of the 3D gene against the more variable VP1 (Reuter et al., 2009; Yu et al., 2011) in which even genetic recombinations have been identified (Jin et al., 2015; Wang et al., 2012) In summary, this work did not demonstrate a clear relationship between the detection of porcine kobuvirus in enteric samples of pigs and diarrhea because infection rate was similar in healthy animals The porcine kobuvirus sequences from Slovakia were unique at a genetic level and they were not related to other European nucleotide sequences in the phylogenetic tree Acknowledgement We would like to thank Peter Nettleton, Edinburgh for critical reading of manuscript and correction of English grammar This work was supported by VEGA project No 1/0342/14, APVV-15-0415, by project INFEKTZOON (ITMS 26229120002) and MediPark Kosice (ITMS 26220220185) from EU References Amimo, J.O., Okoth, E., Junga, J.O., Ogara, W.O., Njahira, M.N., Wang, Q., Vlasova, A.N., Saif, L.J., Djikeng, A., 2014 Molecular detection and genetic 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categories 3.2 Prevalence of rotavirus A in healthy and diarrheic pigs Rotavirus A was detected in of 17 farms The results are summarized in Table Rotavirus infection was... Table Prevalence of porcine group A rotavirus in healthy and diarrheic pigs Age group Suckling Weaned Fattening Total Number 14 6 14 7 12 1 414 Healthy (n = 2 51) Diarrheic (n = 16 3) Total (n = 414 )